Arthropod-borne viruses (arboviruses) face extreme challenges as they adapt to both arthropod and vertebrate hosts. Arboviruses acutely infect humans and a wide range of other vertebrates, but must persistently infect one or two mosquito species for transmission to their next vertebrate host. Due to interhost cycling, arboviruses encounter vast differences in immune responses and RNA interference (RNAi), mediated by small RNAs. In contrast to in vertebrates, RNAi is the main antiviral response in insects, targeting RNA virus genomes for degradation to limit viral replication. Because of the disparate nature of RNAi in vertebrates versus mosquitoes, strategies for arboviruses to evade and subvert these responses should be equally divergent depending on the host. Therefore, we hypothesize that arbovirus:small RNA interplay will vary radically between mosquito and mammalian hosts. As a model arbovirus, we plan to study chikungunya virus (CHIKV), which causes a painful, debilitating illness, for which there is no vaccine or treatment. The main goal o this proposal is to understand CHIKV interaction strategies with disparate host RNAi components and responses. This work aims to advance our understanding of host-specific small RNA interactions with arboviruses, beginning with CHIKV. Recently, we collaborated with the Darnell lab at The Rockefeller University to apply cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago; termed Ago-CLIP) to virus-infected cells. Ago proteins are essential to all RNAi pathways, and are guided by small RNAs to bind to mRNA or viral RNA targets. Using Ago-CLIP, we can physically map small RNA-mediated Ago binding to viral genomes. This technique offers a unique opportunity to examine functional differences in host species' small RNA interactions with arboviruses. In our specific aims, we propose to use Ago-CLIP to map small RNA-mediated Ago binding to the CHIKV genome in both mammalian and mosquito models 1) in vitro and 2) in vivo, with special attention to interactions relevant to mosquito antiviral responses. Our understanding of arbovirus:host interactions is limited, particularly in mosquito vectors, and has been a major impediment to the development of antiviral control strategies. Our aims will allow us to identify critical CHIKV:small RNA interactions, which may uncover novel drug targets. Furthermore, observation of such small RNA interactions will increase our understanding of determinants of tropism and may inform strategies for disrupting arbovirus transmission. On a global level, systemic mapping of small RNA landscapes for arboviruses will yield insights into the interplay between viruses and antiviral defenses in disparate hosts.